Fig 2: Duplex chromogenic ISH in A1. Adora1 (red) + MBP (aqua). Hematoxylin counterstain (light blue). (A) Bright-field image of A1. Insets correspond to panels –i–viii. (B) MBP + cells plotted by cortical layer. Filled circles, co-expression of Adora1 + MBP. Open circles, MBP only. White matter, wm. Scale bar, 250 µm. (Panels i–viii) 100× DIC images of inset locations in panel A. Solid arrows, dual-labeled; gray arrows, nominal Adora1 puncta. Arrowheads, extrasomatic MBP + puncta (black) and Adora1 + puncta (gray). See text for details. Scale bar, 20 µm.

Fig 3: Cell counts in A1 and MG for each marker combination. Histograms reflect the mean number of single- (blue bars) and dual-labeled cells (red bars) for each cell type marker. (A–C) Neuronal subtypes + Adora1 in A1 by cortical layer. (D–E) Glial subtypes + Adora1 in A1. (G–I) Neuronal subtypes + Adora1 in MG by division. (J–L) Glial subtypes + Adora1 in MG. Error bars, standard deviation. Note variations in y-axis scale between panels.

Fig 4: Duplex chromogenic ISH in A1. Adora1 (red) + Gapdh (aqua). Hematoxylin counterstain (pale blue). (A) Adora1/Gapdh expression across layers of A1. Insets correspond to locations of images in panels B–E. Scale bar, 250 µm. (B–E) 100× DIC images of inset locations in panel A. Adora1 + puncta (red dots) are concentrated within the somatic cytoplasm, mainly in Gapdh + cells. Some cells, often glia (asterisk, panel D) contained no puncta. Adora1 + puncta were also located along fine peripheral processes (arrowheads). Scale bar, 20 µm.

Fig 5: Fluorescence FISH in layer 3 of A1, combining Adora1 with neuronal or glial markers. (A) Adora1 (red) with neuronal markers: VGluT1 (white), VGluT2 (yellow), and VGAT (green). (B) Adora1 (red) with glial markers: MBP/oligodendrocytes (white), Aqp4/astrocytes (yellow), and Itgam/microglia (green). Reactive puncta (dots) of different markers do not colocalize, but may be co-expressed within the same cell (e.g., Adora1 + VGluT1). Scale bar, 20 µm.